Expression Of H-Ras Research Articles

The potential mechanism of Neu5Gc inducing colorectal cancer based on network pharmacology and experimental validation.

Colorectal cancer has high morbidity and mortality worldwide, especially in western countries; the incidence of colorectal cancer has been high, which is closely related to the high intake of red meat; and the N-glycolylneuraminic acid (Neu5Gc) is responsible for red meat-induced colorectal cancer. A large number of previous studies have suggested that exogenous Neu5Gc-activated inflammation induced the occurrence of colorectal cancer. However, it has not been known whether the Neu5Gc has a direct inducing effect on colorectal cancer. In this study, we found that Neu5Gc promoted the proliferation of colorectal cancer cells and normal intestinal epithelial cells, and further screened out 98 Neu5Gc targets related to the occurrence and development of colorectal cancer by network pharmacology. Subsequently, GO and KEGG enrichment analyses of these targets revealed that mainly enriched in the PI3K-Akt signaling pathway. Then, we selected SRC, HRAS, CDK2, CCNA2, and AKT2 as core targets based on the phenomena of the previous experiments and the available literature reports, and then we used AutoDock for molecular docking with Neu5Gc; the results found that these five genes could bind to Neu5Gc stably. In vitro experiments showed that the mRNA levels of SRC, HRAS, AKT2, CDK2, and CCNA2 were upregulated and the protein levels of HRAS, AKT2, and CCNA2 were enhanced in FHC and SW620 cells after Neu5Gc (100ng/mL) treatment. In conclusion, this study revealed that Neu5Gc probably acted as a carcinogen that stimulates the expression of proto-oncogene HRAS and the PI3K-Akt pathway and accelerated cell cycle progression. These findings revealed a novel mechanism that Neu5Gc promoted the occurrence and development of colorectal cancer.

Analysis of H-ras Mutations and Immunohistochemistry in Recurrence Cases of High-Grade Oral Squamous Cell Carcinoma.

This study is focused on the identification of gene mutations in H-ras which are probably associated with tumor recurrence in oral squamous cell carcinoma (OSCC) following conventional therapy. Surgically removed biopsies from OSCC patients without recurrence (n = 43) and biopsies from recurrent cases (n = 19) were analyzed. Also, gingival tissues (n = 5) from normal individuals were processed and considered as control. DNA was extracted and amplified using primers for exons 1 and 2 for the H-ras gene, and then DNA products were analyzed using Sanger's sequencing technique. Besides, H-ras expression was compared in samples by immunostaining (IHC), using anti-ras antibody. Demographic data show that smoking habit in patients and recurrent tumors was ~ 44.1 and 78%, respectively. The major site of malignancy was tongue tissue (40-60%). The rate of pathological stage III/IV were 41.8 and 100% in primary tumors and recurrence malignancy respectively. The sequencing data showed that a specific mutation in H-ras gene, Gly12Ala (G6266A) in recurrence samples and primary cases was detected in ~ 66.6% and 10% respectively. Accumulation of H-ras protein in tissues was relatively high scores (> 5) in both primary and recurrence tumors. The H-ras mutation detected was associated with increased level of H-ras protein accumulated in the malignant cells (IHC data). These data may suggest that regardless of the causes and factors involved, Gly12Ala (G6266A) is associated with recurrence in high-grade OSCC tumors.

Modulating myoblast differentiation with RNA-based controllers.

Tunable genetic controllers play a critical role in the engineering of biological systems that respond to environmental and cellular signals. RNA devices, a class of engineered RNA-based controllers, enable tunable gene expression control of target genes in response to molecular effectors. RNA devices have been demonstrated in a number of systems showing proof-of-concept of applying ligand-responsive control over therapeutic activities, including regulation of cell fate decisions such as T cell proliferation and apoptosis. Here, we describe the application of a theophylline-responsive RNA device in a muscle progenitor cell system to control myogenic differentiation. Ribozyme-based RNA switches responsive to theophylline control fluorescent reporter expression in C2C12 myoblasts in a ligand dependent manner. HRAS and JAK1, both anti-differentiation proteins, were incorporated into RNA devices. Finally, we demonstrate that the regulation of HRAS expression via theophylline-responsive RNA devices results in the modulation of myoblast differentiation in a theophylline-dependent manner. Our work highlights the potential for RNA devices to exert drug-responsive, tunable control over cell fate decisions with applications in stem cell therapy and basic stem cell biology research.

Expression of SARS-CoV-2-Related Surface Proteins in Non-Small-Cell Lung Cancer Patients and the Influence of Standard of Care Therapy.

Simple SummarySARS-CoV-2 is a respiratory virus that uses ACE2 for host cell entry and the spike protein is primed by, among others, TMPRSS2 and FURIN. The goal of this study was to determine in which non-small-cell lung cancer (NSCLC) patients these proteins are expressed on the membrane of the lung cancer cells and in which patients this increased ACE2 expression results in higher levels of soluble (s)ACE2 in their serum. In addition, we studied the influence of standard of care (SOC) therapies on sACE2 levels. Membranous (m)ACE2 was co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC patients, and mACE2 and sACE2 were more frequently expressed in mutant EGFR patients but not mutant-KRAS patients. Importantly, systemic SOC therapies did not result in increased sACE2 levels. This indicates that cancer cells can be infected by SARS-CoV-2 in these patients, as well as that soluble ACE2 could impact the course of COVID-19.In this study, we aimed to study the expression of SARS-CoV-2-related surface proteins in non-small-cell lung cancer (NSCLC) cells and identify clinicopathological characteristics that are related to increased membranous (m)ACE2 protein expression and soluble (s)ACE2 levels, with a particular focus on standard of care (SOC) therapies. ACE2 (n = 107), TMPRSS2, and FURIN (n = 38) protein expression was determined by immunohistochemical (IHC) analysis in NSCLC patients. sACE2 levels (n = 64) were determined in the serum of lung cancer patients collected before, during, or after treatment with SOC therapies. Finally, the TCGA lung adenocarcinoma (LUAD) database was consulted to study the expression of ACE2 in EGFR- and KRAS-mutant samples and ACE2 expression was correlated with EGFR/HER, RAS, BRAF, ROS1, ALK, and MET mRNA expression. Membranous (m)ACE2 was found to be co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC samples and limited to the adenocarcinoma subtype. TMPRSS2 showed predominantly atypical cytoplasmic expression. mACE2 and sACE2 were more frequently expressed in mutant EGFR patients, but not mutant-KRAS patients. A significant difference was observed in sACE2 for patients treated with targeted therapies, but not for chemo- and immunotherapy. In the TCGA LUAD cohort, ACE2 expression was significantly higher in EGFR-mutant patients and significantly lower in KRAS-mutant patients. Finally, ACE2 expression was positively correlated with ERBB2-4 and ROS1 expression and inversely correlated with KRAS, NRAS, HRAS, and MET mRNA expression. We identified a role for EGFR pathway activation in the expression of mACE2 in NSCLC cells, associated with increased sACE2 levels in patients. Therefore, it is of great interest to study SARS-CoV-2-infected EGFR-mutated NSCLC patients in greater depth in order to obtain a better understanding of how mACE2, sACE2, and SOC TKIs can affect the course of COVID-19.

Effects of silencing farnesyltransferase on the migration, invasion, and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells.

This study aimed to investigate the effects of farnesyltransferase (FTase) on the migration, invasion, and epithelial-mesenchymal transition (EMT) of SACC-LM and SACC-83 cells in salivary adenoid cystic carcinoma and determine the relative mechanism. Three small interfering RNA (siRNA) sequences were designed and constructed based on the human FTase gene sequence. The SACC-LM and SACC-83 cells in the logarithmic growth period were used, and the expression of FTase was suppressed by liposomal transient transfection. The tested cells were categorized as the FTase-siRNA-1, FTase-siRNA-2, and FTase-siRNA-3 groups. Both negative control group (NC-siRNA) and blank control group (only transfection reagent was added) were set. The mRNA expression of FTase and HRAS was detected by quantitive real-time polymerase chain reaction, and the silencing efficiency was determined. The expression levels of FTase, HRAS, protein kinase B (AKT), phospho-AKT, p65, phospho-p65 (Ser563), E-cadherin, vimentin, matrix metalloproteinase (MMP)-9 protein, and HRAS membrane protein were detected by Western blot. Transwell assay and wound healing assay were used to detect the invasion and migration abilities of cells. The relative expression of FTase mRNA and protein in the FTase-siRNA-1 group decreased compared with those in the control group (P<0.05). HRAS mRNA and total protein expression had no significant difference (P>0.05), and the relative expression of HRAS membrane protein decreased (P<0.05). The relative expression of E-cadherin increased (P<0.05), vimentin decreased (P<0.05), and MMP-9 decreased (P<0.05). There was no significant difference in the relative expression levels of the RAS/PI3K/AKT/nuclear factor-κB signaling pathway-related proteins AKT and p65 (P>0.05), but the relative expression levels of phospho-AKT and phospho-p65 decreased. The invasion and migration ability of the FTase-siRNA-1 group significantly decreased compared with that in the control group (P<0.05). Silencing FTase in vitro could effectively inhibit the invasion and migration of SACC-LM and SACC-83 cells by interfering with the localization of the HRAS membrane protein and regulating the RAS/PI3K/AKT/nuclear factor-κB signaling pathway to mediate EMT.

HRAS expression predicts Lenvatinib responsiveness in human primary gastroenteropancreatic neuroendocrine tumor cells.

e16211 Background: Neuroendocrine neoplasms (NENs) are a rare group of heterogeneous tumors, with clinical presentation ranging from indolent and small lesions to very aggressive, metastatic and therapy refractory forms. Despite a high number of trials, few drugs have been introduced in the clinical practice and therapeutic options for systemic intervention are limited. The recent TALENT trial demonstrated the efficacy of Lenvatinib, a multi-tyrosine kinase inhibitor (MKI), in the treatment of patients with advanced well differentiated gastroenteropancreatic neuroendocrine tumors (GEP-NET) progressing from targeted therapies or somatostatin analogues with no other treatment indication. Lenvatinib might represent a novel therapeutic opportunity for these rare tumor patients. However, the treatment approach with MKI lacks clinically validated tissue or blood biomarkers to guide patient selection and improve efficacy. Methods: Here we established 11 human primary cultures from patients with GEP-NEN of different grades and sites of origin and assessed the antitumor activity of Lenvatinib compared with standard treatment agents. All primary cultures were molecularly characterized to identify possible predictors of response. Results: Lenvatinib exerted a significant inhibition of cell growth in primary GEP-NET cells, with median survival inhibitions similar or higher than those of standard treatments. Of the 11 primary cultures analyzed in our case series, 6 were classified as responder with survival inhibition higher than 15%, and 5 as non-responder with survival inhibition lower than 15%. A high expression of HRAS in the tumor tissues compared to the matched healthy tissues significantly correlated with responsiveness of primary cells to Lenvatinib. All 5 non-responder patients showed low expression of HRAS, while of the 6 responder patients, 5 showed a high HRAS expression and only 1 low. None of the other evaluated clinical variables (grade, Ki67, site of origin and syndromic disease) correlated with response. Conclusions: In conclusion, we believe that the evaluation of HRAS expression and mutation might be of great interest in GEP-NET patients receiving Lenvatinib to improve patient selection.

Microarray analysis of potential biomarkers of brachial plexus avulsion caused neuropathic pain in male rat

The present study aimed to investigate the expression of mRNA in the brachial plexus avulsion neuropathic pain model and analyze biological functions. Microarray mRNA assay and reverse transcriptase quantitative polymerase chain reaction (RT-PCR) were conducted. The whole blood was collected from two groups for Microarray mRNA analysis. The predicted mRNA targets were studied by gene ontology analysis and pathway analysis. We identified 3 targeted mRNAs, including PIK3CB, HRAS, and JUN. The results showed that PIK3CB, HRAS, and JUN gene expression was increased in the control group but decreased in the neuropathic pain group. These findings indicate that certain genes may be important biomarkers for the potential targets for the prevention and treatment of brachial plexus avulsion caused neuropathic pain.

Endothelial-Derived APT1-Mediated Macrophage-Endothelial Cell Interactions Participate in the Development of Atherosclerosis by Regulating the Ras/MAPK Signaling Pathway.

Acyl-protein thioesterase 1 (APT1) can affect H-Ras localization and function by promoting its depalmitoylation. However, relatively little attention has been paid to the effects of APT1 on H-Ras in the cardiovascular system. In this study, we revealed its roles in atherosclerosis development using oxidative low-density lipoprotein (ox-LDL)-induced endothelial dysfunction models and a Western diet-induced ApoE−/− mouse model. The results showed that APT1 expression was up-regulated, while that of miR-138-5p (miR-138) was down-regulated (p < 0.05) in this model. In the meantime, APT1 and H-Ras were translocated from the cytoplasm to the plasma membrane. Bioinformatic analysis and double fluorescence identified miR-138 as the upstream regulator of APT1. APT1 knockdown regulated H-Ras localization and expression, which subsequently affected the MAPK signaling pathway and the expression of its downstream factors. Further research indicated that human umbilical vein endothelial cells (HUVECs)-derived biogenic nanoparticles (BiNPs), hBPs secretion, and RNA expression of hBP-loaded APT1 were increased (p < 0.05) in the ox-LDL induced endothelial dysfunction model. Meanwhile, the HUVECs-derived APT1 could further affect macrophage function through hBP transportation. Altogether, this study demonstrated that the miR-138-APT1 axis may be partially responsible for atherosclerosis development by regulating the H-Ras-MAPK signaling pathway and hBP transportation. The results also shed novel insight on the underlying mechanisms of, and identify potential diagnostic and therapeutic targets for, atherosclerotic cardiovascular diseases in the future.

Identification of Potential Core Genes in Parkinson's Disease Using Bioinformatics Analysis.

Purpose This study aimed to explore new core genes related to the occurrence of Parkinson's disease (PD) and core genes that can lead to the progression of PD. Methods The expression profile data of GSE42966, which contained six substantia nigra tissues isolated from normal individuals and nine substantia nigra tissues isolated from patients with PD, were obtained from Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified, followed by functional enrichment analysis and protein-protein interaction (PPI) network construction. We then identified 10 hub genes and analyzed their expression in different Braak stages. Results A total of 773 DEGs were identified that were significantly enriched in metabolic pathways. Ten hub genes were identified through the PPI network, namely, GNG3, MAPK1, FPR1, ATP5B, GNG2, PRKACA, HRAS, HSPA8, PSAP, and GABBR2. The expression of HRAS was different in patients with PD with Braak stages 3 and 4. Conclusion These 10 hub genes and the metabolic pathways they are enriched in may be involved in the pathogenesis of PD. HRAS may have potential value in predicting the progression of PD.

DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway.

To determine the differentially expressed proteins (DEPs) between paired samples of cervical cancer (CC) and paracancerous tissue by quantitative proteomics and to examine the effects of DUSP7 expression on the tumorigenesis and progression of CC. Proteomic profiles of three paired samples of CC and paracancerous tissue were quantitatively analysed to identify DEPs. The relationship between DEP expression and patient clinicopathological characteristics and prognosis was evaluated. The effects of the selected DEPs on CC progression were examined in SIHA cells. A total of 129 DEPs were found. Western blot and immunohistochemistry (IHC) staining analyses confirmed the results from quantitative proteomic analysis showing that the selected DEP, HRAS, P‐ERK1/2, and PLD1 levels were increased, whereas the DUSP7 level was decreased in CC tissue compared with the paired normal paracancerous tissues. The IHC results from the CC TMA analysis showed that the decreased expression of DUSP7 (p = 0.045 and 0.044) was significantly associated with a tumour size >2 cm and parametrial infiltration. In addition, the decreased expression of DUSP7 and increased expression of p‐ERK1/2 were adversely related to patient relapse (p = 0.003 and 0.001) and survival (p = 0.034 and 0.006). The expression of HRAS and p‐ERK1/2 was decreased in DUSP7‐SIHA cells compared with NC‐SIHA cells (p = 0.0003 and 0.0026). Biological functions in vitro, including invasion, migration and proliferation and tumour formation in vivo were decreased in DUSP7‐SIHA cells (all p < 0.05) but increased in shDUSP7‐SIHA cells (all p < 0.05). DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway.

Exploration of the Potential Mechanism of Tao Hong Si Wu Decoction for the Treatment of Breast Cancer Based on Network Pharmacology and In Vitro Experimental Verification.

BackgroundTao Hong Si Wu Decoction (THSWD) is a well-known traditional Chinese medicine used clinically alone or combined with drugs to treat breast cancer. However, there has been no study to date on the underlying mechanisms of its therapeutic effects.ObjectivesTo explore the potential mechanism of THSWD for the treatment of breast cancer using network pharmacology and experimental research.MethodsThe active ingredients of THSWD were screened according to Lipinski’s rule of five based on the 107 ingredients of THSWD identified by UPLC-Q-TOF-MSE. The targets of THSWD and breast cancer from multiple databases were collected, and a Compound-Target-Pathway network based on protein-protein interaction (PPI) was constructed. Gene ontology (GO) analysis and KEGG pathway analysis were performed via the DAVID server. Molecular docking studies verified the selected key ingredients and key targets. The results of network pharmacology were verified by in vitro experiments. Including the effects of THSWD drug-containing rat serum (THSWD serum) on cell proliferation, and on the targets HRAS, MAPK1, AKT1, GRB2, and MAPK14 were assayed by RT-qPCR and Western blot assays.ResultsIn total, 27 active ingredients including 8 core components, were obtained from 107 ingredients and 218 THSWD target genes for the treatment of breast cancer were identified. THSWD is active in the treatment of breast cancer by targeting Ras, FoxO, PI3K-Akt and other signaling pathways. MCF-7 and MDA-MB-231 cell proliferation was inhibited by THSWD serum in a time and concentration dependent manner. THSWD could regulated the RNA and protein expression of core targets HRAS, MAPK1, AKT1, GRB2, and MAPK14 for treatment of breast cancer.ConclusionThe results of network pharmacology study showed that THSWD is active against breast cancer by intervening with multiple targets and pathways. Luteolin, kaempferol, senkyunolide E, and other 8 compounds may be the core active ingredients of THSWD in the treatment of breast cancer. THSWD treatment of breast cancer may be related to targeting Ras, FoxO, PI3K-Akt, and other signal pathways associated with the core targets HRAS, MAPK1, AKT1, GRB2, and MAPK14.

Expression of oncogenic HRAS in human Rh28 and RMS-YM rhabdomyosarcoma cells leads to oncogene-induced senescence

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level—eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a “quiet” genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.

Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-RasG12V) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-RasG12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-RasG12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-RasG12V, which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

Identification and functional analysis of a novel phospholipase D2 gene mutation associated with familial systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a kind of autoimmune inflammatory connective tissue disease which seriously endangers human health. Genetic factors play a key role in the pathogenesis of SLE. This study aims to investigate a novel phospholipase D2 (PLD2) mutation associated with familial SLE, and further explore the underlying mechanism of the mutation in SLE. The blood samples from a SLE patient, the patient's parents, and 147 normal controls were collected and DNA was extracted. Whole genome high-throughput sequencing was performed in the patient and her parents and the results were further analyzed by various bioinformatics methods. The wild type (wt), mutant type (mu), and negative control PLD2 plasmids were further constructed and transfected into 293 cells. The expression level of HRAS protein in 293 cells was detected by Western blotting. In this SLE family, the female SLE patient and her mother, 1 in generation II and 1 in generation III had typical clinical manifestations of SLE, and all of them had lupus nephritis at early stage. The genetic characteristics are consistent with autosomal dominant inheritance. A novel PLD2 heterozygous mutation (c.2722C>T) was found in the patient and her mother, but not in her father and other normal controls. Compared with wtPLD2 plasmid and negative control PLD2 plasmid, the expression of HRAS in 293 cells transfected with muPLD2 plasmid was significantly up-regulated (both P<0.05). PLD2 c.2722C>T mutation may be one of the pathogeny of SLE in this family.

Clinical significance of HRAS and KRAS genes expression in patients with non\u2013small-cell lung cancer - preliminary findings

BackgroundThe RAS family protooncogenes, including KRAS, NRAS and HRAS, encode proteins responsible for the regulation of growth, differentiation and survival of many cell types. The HRAS and KRAS oncogene mutations are well defined, however, the clinical significance of RAS expressions in non–small-cell lung cancer (NSCLC) is still uncertain.MethodsA total of 39 whole blood samples of NSCLC (the investigated group), collected at three points of time: at the time of diagnosis, 100 days and 1 year after the surgery as well as 35 tissue samples obtained during the surgery were included in this study. HRAS and KRAS genes mRNA expression were assessed using quantitative real-time polymerase chain reaction techniques.ResultsIncreased relative HRAS mRNA level in blood was found significantly more frequently in the group of smokers (p = 0.008). Patients with squamous cell carcinoma subtypes of NSCLC were more likely to show an overexpression of HRAS gene in blood, but not statistically significant (p = 0.065). In tumor tissue overexpression of HRAS gene was associated with adenocarcinoma subtype (p = 0.049). No statistically significant associations were found for the expression of KRAS with any clinicopathological parameters, except the age of patients, within the study. There were no differences between the relative HRAS and KRAS genes expression levels in blood samples taken from the same patients during the 3 observation points, as well as between blood collected from patients before surgery and tissue samples obtained during operation.ConclusionThe potential associations between high HRAS expression levels, age, smoking status and histological type of cancer were observed, which emphasizes the need for further study of the RAS family. Therefore, subsequent research involving larger numbers of patients and a longer follow-up, as well as multicenter study are necessary to confirm our findings.

Epigenetic regulation of TGF-\u03b2-induced EMT by JMJD3/KDM6B histone H3K27 demethylase

Transforming growth factor-β (TGF-β) signaling pathways are well-recognized for their role in proliferation and epithelial–mesenchymal transition (EMT) of cancer cells, but much less is understood about their contribution to interactions with other signaling events. Recent studies have indicated that crosstalk between TGF-β and Ras signaling makes a contribution to TGF-β-mediated EMT. Here, we demonstrate that Jumonji domain containing-3 (JMJD3 also called KDM6B) promotes TGF-β-mediated Smad activation and EMT in Ras-activated lung cancer cells. JMJD3 in lung cancer patients was significantly increased and JMJD3 expression in lung tumor tissues was correlated with expression of K-Ras or H-Ras in particular, and its expression was regulated by Ras activity in lung cancer cells. JMJD3 promotes TGF-β-induced Smad activation and EMT in Ras-activated lung cancer cells through the induction of syntenin, a protein that regulates TGF-β receptor activation upon ligand binding. Tissue array and ChIP analysis revealed that JMJD3 epigenetically induces syntenin expression by directly regulating H3K27 methylation levels. Mechanical exploration identified a physical and functional association of JMJD3 with syntenin presiding over the TGF-β in Ras-activated lung cancer cells. Taken together, these findings provide new insight into the mechanisms by which JMJD3 promotes syntenin expression resulting in oncogenic Ras cooperation with TGF-β to promote EMT.

Knockdown of the DJ-1 (PARK7) gene sensitizes pancreatic cancer to erlotinib inhibition

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib, in combination with gemcitabine, has been shown to be a promising therapy in the treatment of pancreatic cancer. Our previous study showed that DJ-1 promotes invasion and metastasis of pancreatic cancer cells by activating SRC/extracellular signal-regulated kinase (ERK)/uPA. The aim of this study was to evaluate whether knockdown of DJ-1 expression can sensitize pancreatic cancer cells to erlotinib treatment. Knockdown of DJ-1 expression accelerated erlotinib-induced cell apoptosis and improved the inhibitory effect of erlotinib on pancreatic cancer cell proliferation (for the BxPC-3, PANC-1, and MiaPACa-2 cell lines, regardless of KRAS mutation status) in vitro and in xenograft tumor growth in vivo. Knockdown of DJ-1 decreased K-RAS expression, membrane translocation, and activity in BxPC-3 cells. Knockdown of DJ-1 also decreased K-RAS, H-RAS, and N-RAS expression in PANC-1 and MiaPACa-2 cells. Knockdown of DJ-1 synergistically inhibited AKT and ERK1/2 phosphorylation with erlotinib in pancreatic cancer cells. These findings indicate that DJ-1 may activate the RAS pathway, reinforcing erlotinib drug resistance. Therefore, blocking DJ-1 in combination with the EGFR tyrosine kinase inhibitor erlotinib may be an attractive therapeutic target in pancreatic cancer.

Circ_0045714 alleviates TNF-α-induced chondrocyte injury and extracellular matrix degradation through miR-218-5p/HRAS axis.

Emerging evidence suggests that dysregulated circular RNAs (circRNAs) play a pivotal role in osteoarthritis (OA). Circ_0045714 is a functional circRNAs, and has been revealed to involve in the process of OA. However, the molecular mechanisms by which circ_0045714 regulates OA progression are not thoroughly elucidated. Circ_0045714 expression was decreased in OA and TNF-α-induced chondrocytes, ectopic overexpression of circ_0045714 abolished TNF-α-induced cell apoptosis, inflammation, extracellular matrix (ECM) degradation promotion and proliferation inhibition. In a mechanical study, circ_0045714 targeted miR-218-5p, and miR-218-5p overexpression reversed the effects of circ_0045714 on TNF-α-induced chondrocytes. Besides that, HRAS was a target of miR-218-5p, and HRAS knockdown attenuated the protective effects of miR-218-5p inhibition on TNF-α-induced chondrocyte dysfunction. Additionally, circ_0045714 could regulate HRAS expression via miR-218-5p in chondrocytes. Up-regulation of circ_0045714 suppressed TNF-α-induced chondrocyte growth inhibition, inflammation, and ECM degradation via miR-218-5p/HRAS axis, suggesting a novel insight into the pathogenesis of OA and the potential protective role of circ_0045714 in the occurrence and development of OA.

Effect of Ras-guanine nucleotide release factor 1-mediated H-Ras/ERK signaling pathway on glioma

ObjectiveTo investigate the function of Ras-guanine nucleotide release factor 1 (Ras-GRF1) in glioma through mediating H-Ras/ERK signaling pathway. MethodsRas-GRF1, H-Ras, K-Ras and N-Ras expressions in glioma and normal brain tissues were detected via Immunohistochemistry. Glioma cells (U87 cells, U251 cells and primary human glioma cells) were transfected with Ras-GRF1 siRNA, H-Ras siRNA and/or Ras-GRF1 lentivirus activation particles. Then, the following aspects were evaluated: cell proliferation by MTT assay, clonogenic ability by the plate clone formation experiment, cell migration and invasion by Wound-healing and Transwell assays, and cell apoptosis by Annexin-V-FITC/PI staining. The protein expressions were measured by Western blotting. Subcutaneous and orthotopic mouse models of glioma were conducted to determine the role of Ras-GRF1 in glioma tumorigenesis. ResultsRas-GRF1, H-Ras, K-Ras and N-Ras expressions were upregulated in the glioma tissues, which were correlated with the WHO grade of glioma. Besides, Ras-GRF1 expression was positively related to H-Ras expression. Ras-GRF1 siRNA could reduce the expression of H-Ras and p-ERK/ERK in glioma cell. H-Ras siRNA inhibited the proliferation, clone formation, migration and invasion, and enhance the apoptosis of glioma cells, which, however, were reversed by Ras-GRF1 lentivirus activation particles. In vivo experiments also revealed that Ras-GRF1 shRNA reduced the volume and weight of the tumors in the nude mice, with down-regulations of H-Ras and p-ERK/ERK. ConclusionRas-GRF1 was upregulated in glioma tissues and correlated with its malignancy and prognosis. Silencing Ras-GRF1, through mediating H-Ras/ERK pathway, may suppress the growth and metastasis of glioma.

Effect of photodynamic therapy on expression of HRAS, NRAS and caspase 3 genes at mRNA levels, apoptosis of head and neck squamous cell carcinoma cell line.

This study aimed to assess the effect of photodynamic therapy (PDT) on expression of CASP3, NRAS and HRAS genes at mRNA levels, and apoptosis of head and neck squamous cell carcinoma (HNSCC) cell line. In order to complete the present in vitro study, HNSCC cell line (NCBI C196 HN5) purchased from Pasteur Institute. Cells were divided into four groups; Group 1: photodynamic treatment (laser + methylene blue (MB) as photosensitizer), group 2: MB, group 3: laser (with 660 nm wavelength), and group 4: control (without any treatment). To determine the optimal concentration of MB, in a pilot study, toxicity of MB in different concentration was assessed using MTT assay. Cells in group 1, 2 and 3 was treated at optimal concentration of MB (1.6 μg/mL). Gene expression at mRNA levels was assessed after 24 h incubation, using real-time (qRT)-PCR. The expression of BAX and BCL2 genes at the mRNA levels was analyzed to evaluate apoptosis. 2-ΔΔCt values of BCL2, BAX, CASP3, NRAS, and HRAS in groups was analyzed using ANOVA. Tukey's HSD and Games Howell test was used to compare between two groups. Over-expression of BAX (p < 0.001), CASP3 (p < 0.001) and down-regulation of BCL2 (p = 0.004), HRAS (p = 0.023) and NRAS (p = 0.045) were noted in group 1 (PDT), compared with the control group. Treatment by laser alone induce down-regulation of CASP3 (p < 0.05), BAX (p < 0.05), BCL2 (p > 0.05), HRAS (p > 0.05) and NRAS (p > 0.05). PDT caused down-regulation of NRAS, HRAS and BCL2 and over-expression of CASP3 and BAX genes at mRNA levels in HNSCC cell line. The present study raises the possibility that the role of MB on BCL2 down-regulation and BAX and CASP3 over-expression was higher than laser alone while it seems that laser alone was more effective than MB in HRAS and NRAS down-regulation.